CN105670962B - The efficient degrading bacteria of one plant of aromatic hydrocarbon and its application - Google Patents

The efficient degrading bacteria of one plant of aromatic hydrocarbon and its application Download PDF

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CN105670962B
CN105670962B CN201610045801.9A CN201610045801A CN105670962B CN 105670962 B CN105670962 B CN 105670962B CN 201610045801 A CN201610045801 A CN 201610045801A CN 105670962 B CN105670962 B CN 105670962B
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benzene
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toluene
styrene
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冯守帅
杨海麟
张燕可
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Jiangnan University
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/34—Chemical or biological purification of waste gases
    • B01D53/46—Removing components of defined structure
    • B01D53/72—Organic compounds not provided for in groups B01D53/48 - B01D53/70, e.g. hydrocarbons
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    • B01D53/84—Biological processes
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    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract

The invention discloses the efficient degrading bacteria of one plant of aromatic hydrocarbon and its applications, belong to environment pollutant biological treatment technical field.The fine bacterium SS-2 of fibrosis fiber of the invention, deposit number are as follows: CCTCC NO:M 2016031.The bacterial strain all has preferable degradation capability to benzene, toluene, styrene, illustrates that the bacterial strain has wider substrate Utilization ability and stronger organic solvent tolerance.With the growth state cell of the bacterium at the same time containing benzene, toluene, styrene minimal medium in culture find that the bacterium can be in higher benzene class substrate concentration range using it as carbon source fast-growth, this also illustrates the bacterial strain with stronger organic solvent tolerance from another point of view.The bacterium is suitable for the degradation of benezene material in industrial wastewater exhaust gas, to improve the efficiency of the existing biological treatment substance.

Description

The efficient degrading bacteria of one plant of aromatic hydrocarbon and its application
Technical field
The present invention relates to the efficient degrading bacteria of one plant of aromatic hydrocarbon and its applications in terms of mono-cyclic aromatic alkane degradation, belong to ring Border pollutant biologic treating technique field.
Background technique
With the fast development of economic society, the continuous improvement of living standard, people's lives state is no longer solution Certainly basic problem of food and clothing and material requisite, but it is changed into the craving to the living environment of high-quality and safety, problem of environmental pollution Also it is increasingly valued by people, researcher also has been devoted to microorganisms to the mechanism of degradation of arene pollutant The research of reason.Some compound fragrant hydrocarbons can bring larger threat to the safe and healthy of people, such as benzene, toluene, styrene energy Enough Central nervous systems generate paralysis effect, cause acute poisoning, and to the irritating effect of skin and mucosa, wherein benzene has been bright True easy carcinogenic substance.Currently, method of the processing containing volatile aromatic hydrocarbon waste gases mainly has combustion method, absorption method, absorption process, cold Solidifying method, bioanalysis.Wherein combustion method has higher treatment effeciency for the organic exhaust gas of processing low concentration and high concentration；It absorbs The efficiency of the organic exhaust gas of method, condensation method processing high concentration is higher；And absorption method and biological rule are suitable for processing low concentration and have Machine exhaust gas.The some organic matters of combustion method treatment process may not be able to convert completely, generate toxic intermediate product；The fortune of absorption method Row is at high cost, and absorbing liquid still needs to be handled again；Absorption process needs to provide very high pressure, and absorbing liquid needs to recycle； The process is more complicated for condensation method, and the entrance of sample requires stringent.Bioanalysis is the own metabolism using microorganism, passes through micro- life The intracorporal enzymatic catalysis of object is broken phenyl ring, is further metabolized as other intermediate products, is then degraded by microorganisms as titanium dioxide Carbon and water.For the improvement of the aromatic hydrocarbon of volume low-concentration big in the industries such as petrochemical industry, bioanalysis is compared with other methods, There is biggish superiority in terms of economic and technical.The simple process and low cost of Biochemical method volatile organic compounds It is honest and clean and without secondary pollution, it is used widely in some countries.But the technology still has in practical applications much asks Topic, one of problem are that the organic exhaust gas effect of Biochemical method low concentration is preferable, but handles the exhaust gas effect of high concentration But very low.
Therefore, screen one plant of organic solvent-resistant and can efficient degradation aromatic hydrocarbon mono-cyclic aromatic surfactant hydrocarbon degradation bacteria, for into one Step promote bioanalysis degradation mononuclear aromatic using particularly important.
Summary of the invention
For the main object of the present invention aiming at the problems and shortcomings present on, providing one plant can efficient degradation monocycle virtue The bacterial strain of fragrant hydrocarbon makes it be applied to the processing of petrochemical wastewater exhaust gas.
There is provided the bacterial strain of one plant of energy efficient degradation mononuclear aromatic, the bacterial strains for the first problem that the present invention solves It is named as the fine bacterium of fibrosis fiber (Cellulosimicrobium cellulans) SS-2.
Sewage disposal system of the bacterial strain screening from certain company is as obtained from building rapid screening method.
Preferably, the GenBank accession number of the 16S rRNA gene order of affiliated bacterial strain is KU170621, with Cellulosimicrobium cellulans B107 (GenBank:HQ877779.1) has 100% similitude.
Preferably, bacterial strain provided by the present invention, belongs to gram-positive bacteria, is yellow, surface light on LB culture medium Sliding wet rule, neat in edge, protrusion is opaque, and viscosity is stronger.The bacterial strain can be grown by carbon source of mononuclear aromatics.
The screening technique of the bacterial strain: sewage disposal system sample, using added with benzene, toluene, styrene inorganic salts Culture medium domestication culture, takes culture solution 50uL to be spread evenly across the training of plate acclimation and screening in the shaking flask for finally taming completion several times Base culture is supported, the preferable bacterium colony scribing line separation of the individual and growing way on plating medium is picked out, is trained in plate acclimation and screening It supports to transfer 3 times on base and obtains purifying bacterial strain.
The suitable growth temperature of the bacterial strain is 25-30 DEG C, can be grown in waste water of the simulation containing mononuclear aromatic, and energy It degrades aromatic hydrocarbon therein.
There is provided the fine bacterium of fibrosis fiber described in one plant for another technical problem that the present invention solves Application of (Cellulosimicrobium cellulans) SS-2 in terms of mono-cyclic aromatic alkane degradation.
The application is in one embodiment of the invention by logarithmic growth phase (i.e. growth state cell) Cellulosimicrobium cellulans SS-2 is inoculated in the inorganic of compound containing benzene (i.e. benzene, toluene, styrene) It is cultivated in salt culture medium, the mononuclear aromatic in culture medium of degrading.The culture, a kind of embodiment in the present invention In, refer to that in temperature be 30 DEG C, pH 7.0, revolving speed is 180r/min shaken cultivation 40h or more.
The fine bacterium Cellulosimicrobium cellulans SS-2 of fibrosis fiber provided by the invention, with nearly ten Year newfound benezene material degradation bacteria strains of document report are compared, and have the advantages that the fine bacterium of (1) fibrosis fiber Benzene, toluene, styrene in Cellulosimicrobium cellulans SS-2 energy degrading waste water；(2) to benzene, toluene, benzene Ethylene degradation efficiency with higher, when concentration of substrate is about 30mg/L, the degradation rate of 48h is up to 86% or more；(3) can It is resistant to the mononuclear aromatic of higher concentration, remains to grow in the mixed liquor of the mononuclear aromatic of high concentration.
Biomaterial preservation
The fine bacterium Cellulosimicrobium cellulans SS-2 of fibrosis fiber, taxology are named as fibrosis The fine bacterium Cellulosimicrobium cellulans SS-2 of fiber is preserved in Chinese Typical Representative culture on January 11st, 2016 Object collection (CCTCC), preservation address are Wuhan, China university, and deposit number is CCTCC NO:M 2016031.
Detailed description of the invention
Fig. 1: the fine bacterium SS-2 of the fibrosis fiber cellular morphology under transmission electron microscope.
Fig. 2: the fine bacterium SS-2 of the fibrosis fiber degradation curve in the minimal medium of the toluene containing various concentration.
Fig. 3: the fine bacterium SS-2 of the fibrosis fiber degradation curve in the minimal medium of the styrene containing various concentration.
Fig. 4: the fine bacterium SS-2 of the fibrosis fiber degradation curve in the minimal medium of the benzene containing various concentration.
Fig. 5: the fine bacterium SS-2 of fibrosis fiber benzene containing 30mg/L, toluene, styrene minimal medium in drop Solution curve.
Specific embodiment
Embodiment 1: bacterial strain acclimation and screening
The sewage 2ml in petroleum waste water processing system is taken to be added in the 250ml conical flask of the minimal medium containing 50ml, it The 5 μ l of mixed liquor (benzene, toluene, concentration of styrene ratio are 8:10:5) of benzene, toluene, styrene is added afterwards, sealing bottleneck (prevents benzene The volatilization of substance), at 30 DEG C, shaking table culture 2d under the conditions of 180r/min.Then fresh nothing is transferred to 5% inoculum concentration Continue in machine salt culture medium domestication (the benezene material content in culture medium successively rises, in shaking flask benzene class mixed liquor additive amount according to It is secondary be 5 μ l, 15 μ l, 25 μ l, 40 μ l, 50 μ l, 60 μ l, 75 μ l, 90 μ l, 100 μ l).(there is bacterium colony to grow after the completion of 6th domestication Then continue to improve domestication concentration), the bacterium solution 50uL after taking domestication every time is spread evenly across plate acclimation and screening culture medium culture 4d, It is selected on plating medium individually and the preferable bacterium colony of growing way --- scribing line separation, plating medium is transferred 3 times and obtained Pure bacterium.
Embodiment 2: bacterial strain identification
Using bacterium full-length genome Rapid extraction kit, the full-length genome of bacterial strain pure culture is extracted, and passes through 16S RDNA universal primer carries out PCR amplification, and PCR amplification system is as follows: 50 μ lPCR reaction systems contain 39.5 μ l of distilled water, 5 μ l's 10 times of buffers, the dNTP of 1 μ l, upstream primer and each 1.25 international unit of 1 μ l, Taq archaeal dna polymerase of downstream primer, genome 2 μ l of template.Amplified production is sequenced, identifying the bacterial strain by alignment is the fine bacterium of fibrosis fiber, is named as The fine bacterium SS-2 of fibrosis fiber (Cellulosimicrobium cellulans SS-2).
Embodiment 3: bacterial strain physiological and biochemical research
Using form such as Fig. 1 of the above-mentioned SS-2 of transmission electron microscope observing.The bacterium is rod-shaped as seen from the figure, there is pod membrane, atrichia. The bacterium can preferably grow at 25-30 DEG C.
By carrying out Gram's staining, capsule stain, methyl red test, V-P test, catalase test etc. to the bacterial strain Physiological and biochemical test determine the bacterium be Gram-positive, atrichia, have pod membrane, methyl red test be it is positive, V-P test is yin Property, catalase be the positive.
Embodiment 4: application of the growth state cell strain in terms of single mononuclear aromatic degradation of substrates
The fine bacterium SS-2 of fibrosis fiber in logarithmic growth phase is inoculated by 2% inoculum concentration containing single aromatic hydrocarbon In the minimal medium of the gradient concentration of substrate (respectively benzene, toluene, styrene), concentration is respectively 30mg/L, 60mg/ L, 90mg/L, controlling initial pH is 7.0, at 30 DEG C, 180r/min shaking table culture 48h, takes a sample every 8h, does headspace gas The content of benzene, toluene and styrene in chromatography survey culture solution.
Wherein minimal medium forms are as follows: 25mL phosphate buffer (K2HPO462g, NaH2PO433g, distilled water 1000mL, adjusts pH to 7.5), NH4NO31g, MgSO4·7H2O 0.5g, CaCl2·2H2O 0.1g, 1mL trace element solution (FeSO4·7H2O 4.5g, ZnSO4·7H2O 0.148g, MnSO4·H2O 0.258g, CoCl2·6H2O0.024g, NiSO4· 7H2O 0.022g, CuCl20.013g, Na2MoO4·2H2O 0.10g, H3BO30.062g, distilled water 1000mL), distilled water 1000mL, 121 DEG C of high pressure steam sterilization 20min, solid add 2% agar.As needed with liquid-transfering gun be added appropriate benzene, toluene, Styrene mixture.
The fine bacterium SS-2 of fibrosis fiber has preferable degradation effect to single benzene-like compounds, 30mg/L, When the concentration of the initial toluene of 60mg/L, 90mg/L, the degradation rate of the bacterium is respectively 85%, 80.8%, 81.7%.In 30mg/ L, under the styrene gradient concentration of 60mg/L, 90mg/L, degradation rate is respectively 88.7%, 80.8%, 83.8%.In the gradient Under benzene substrate in, the degradation rate of the bacterium is 74.7%, 78%, 79%.
The growth state cell of the bacterial strain benzene containing various concentration, toluene, styrene minimal medium in degradation feelings Condition is as shown in figure Fig. 2, Fig. 3 and Fig. 4.
Embodiment 5: application of the growth state cell strain in terms of benzene, toluene, styrene mixed substrates degradation
The fine bacterium SS-2 of fibrosis fiber in logarithmic growth phase is inoculated by 2% inoculum concentration containing 30mg/L's Benzene, toluene, styrene mixed substrates minimal medium in, controlling initial pH is 7.0, at 30 DEG C, the training of 180r/min shaking table Support 48h, take a sample every 8h, do headspace gas chromatography survey culture solution in benzene, toluene and styrene content.
The fine bacterium SS-2 of fibrosis fiber has stronger degradation capability to benzene-like compounds, when benzene, toluene, styrene are mixed Close substrate initial concentration be 30mg/L when, bacterial strain in 48h to the degradation of three kinds of aromatic compounds reach 86% with On.
Benzene class degradation situation such as Fig. 5 institute of the growth state cell of the bacterial strain in the minimal medium of containing benezene compound Show.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that can still make Various modifications and alterations are without departing from the spirit and scope of the invention.Therefore, the description and the appended drawings should be considered as illustrative And not restrictive.

Claims (2)

1. a kind of utilize the fine bacterium of fibrosis fiber (Cellulosimicrobium cellulans) SS-2 degradation mono-cyclic aromatic The method of hydrocarbon, which is characterized in that the fine bacterium SS-2 of fibrosis fiber is inoculated by 2% inoculum concentration containing single aromatic hydrocarbon bottom In the minimal medium of object, single aromatic hydrocarbon concentration of substrate is 30-90mg/L, and controlling initial pH is 7.0, at 30 DEG C, 180r/ Min shaking table culture 48h；The minimal medium composition are as follows: 25mL phosphate buffer, NH4NO31g, MgSO4·7H2O 0.5g, CaCl2·2H2O 0.1g, 1mL trace element solution, distilled water 1 000mL, 121 DEG C of high pressure steam sterilization 20min, root According to needing that mononuclear aromatic is added；The phosphate buffer composition are as follows: K2HPO462g, NaH2PO433g, distilled water 1 000mL adjusts pH to 7.5；The trace element solution composition are as follows: FeSO4·7H2O 4.5g, ZnSO4·7H2O 0.148g, MnSO4·H2O 0.258g, CoCl2·6H2O 0.024g, NiSO4·7H2O 0.022g, CuCl20.013g, Na2MoO4· 2H2O 0.10g, H3BO30.062g, 1 000mL of distilled water；Fine bacterium (the Cellulosimicrobium of fibrosis fiber Cellulans) SS-2, is preserved in China typical culture collection center on January 11st, 2016, and preservation address is that Wuhan, China is big It learns, deposit number is CCTCC NO:M 2016031.
2. the method according to claim 1, wherein the mononuclear aromatic is benzene, toluene or styrene.
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CN108130288B (en) * 2017-12-15 2020-10-09 浙江工业大学 Rhodococcus ruber and application thereof in degrading organic pollutants
CN110721572B (en) * 2019-10-24 2022-04-29 深圳如帆科技有限公司 Preparation method of extract for degrading triphenyl and triphenyl purifying agent
CN111440747B (en) * 2020-05-08 2020-12-01 微米环创生物科技(北京)有限公司 Compound microbial agent for sewage treatment, microecological preparation and application
CN115197864B (en) * 2021-12-31 2023-12-19 广州畅培优食品研究有限公司 Pseudomonas and method for degrading monocyclic aromatic hydrocarbon by using pseudomonas

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